Corticotropin-releasing factor levels in the peripheral plasma and hypothalamus of the rat vary in parallel with changes in the pituitary-adrenal axis.

Impressive evidence has emerged indicating that immunoassayable and bioassayable CRF, which is immunoneutralizable, is present not only in the hypothalamus but in many peripheral tissues as well. Using highly specific and sensitive RIAs and immunoaffinity chromatography to investigate whether this extrabrain CRF circulates in the rat, we found low but clearly measurable levels in peripheral plasma (mean, 11.4 +/- 0.8 pg/ml). Immunological findings were corroborated by fast protein liquid chromatography, which resolves peptides by both hydrophobicity and ionic charge. With this approach the major immunoreactive peak was eluted at the position of synthetic rat CRF standard. To assess whether levels of peripheral plasma CRF-like immunoreactivity (CRF-LI) vary in parallel with those of hypothalamic CRF-LI, we performed studies with low and high dose dexamethasone administration and withdrawal, adrenalectomy, and hypophysectomy. Seven days after oral administration of dexamethasone, there was a decrement in the levels of peripheral plasma and hypothalamic CRF-LI. Depending on the dose, recovery was also found 7 days after cessation of the treatment. After either adrenalectomy or hypophysectomy, there were increments in the levels of CRF-LI in both peripheral plasma and hypothalamus. Thus, concentrations of CRF-LI in the peripheral plasma and in the hypothalamus vary in parallel in response to alterations in the pituitary-adrenal axis.

Levels of corticotropin releasing factor-like immunoreactivity in mammalian hypothalamic and extrahypothalamic brain tissue as determined with a monoclonal antibody to the ovine material.

A monoclonal antibody to ovine corticotropin releasing factor (CRF) has been produced by fusion of a non-producing plasmacytoma cell line P3U1 with spleen cells of Balb/c mice immunized with the synthetic 41 amino acid peptide coupled covalently with rabbit myosin by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. A total immunizing dose of 500 micrograms resulted in a highly specific, high-affinity antibody with a Ka of 0.15 x 10(12) M-1, which was used to establish a specific RIA with a sensitivity of 10 pg/tube. Levels of corticotropin releasing factor-like immunoreactivity (CRF-LI) in a pg/mg of hypothalamic tissue ranged from 4-10 in ovine, 2.5-8 in bovine, 47.5-67.5 in mouse and 2.3-20 in human tissue. Moreover, CRF-LI was widely distributed in extrahypothalamic mouse brain at concentrations approximately one half those seen in hypothalamus.

Corticotrophin-releasing factor in extra-hypothalamic brain of the mouse: demonstration by immunoassay and immunoneutralization of bioassayable activity.

Corticotrophin-releasing factor (CRF) bioactivity has been described in the extra-hypothalamic brain, but its relationship to hypothalamic CRF has remained questionable. Of the seven regions of the mouse brain examined, highest concentrations of CRF-like immunoreactivity (CRF-LI) and bioassayable CRF activity were present in the median eminence and hypothalamus. However, substantial CRF-LI and bioassayable CRF activity were also seen in brain extracts from the amygdala, thalamus, frontal cortex, pons medulla and cerebellum. Bioactivity was largely neutralized by prior incubation with heat-inactivated antiserum to ovine CRF. These findings, in conjunction with previous immunocytochemical evidence, strongly suggest that a substance closely resembling hypothalamic CRF is present in the extrahypothalamic brain of the mouse.

Comparison of methaqualone excretion patterns using Abuscreen ONLINE and EMIT II immunoassays and GC/MS.

A study was performed to compare the ONLINE and EMIT II immunoassays with gas chromatographic/mass spectrometric (GC/MS) analysis of methaqualone metabolites on urine using samples obtained from a clinical study. Urine was collected over a 72 h period from six healthy adults (4 male, 2 female) after oral dosing with 200 mg methaqualone (MTQ). Each urine sample was analyzed by ONLINE and EMIT II. The samples were then analyzed by GC/MS, hydrolyzed with beta-glucuronidase and again analyzed by GC/MS. Both immunoassays showed greater than 600 ng/ml concentrations of drug in each sample by the second void and remained highly positive for the rest of the 72 h. Unhydrolyzed samples analyzed by GC/MS showed both low concentrations of MTQ as well as its five major hydroxylated metabolites. The hydrolyzed samples analyzed by GC/MS showed high concentrations of the hydroxylated metabolites with the 2'-hydroxy and 3'-hydroxy metabolites being present at the highest concentrations, the 4'-hydroxy metabolite at a lower amount and the 6-hydroxy and 2-hydroxy metabolites at the lowest concentrations. The GC/MS data coupled with the antibody cross-reactivity data indicate that the major species in clinical samples that cross-react in both immunoassays are the conjugated forms of the hydroxylated metabolites of MTQ. Therefore when confirming by GC/MS after an immunoassay screen it would be prudent to confirm for the major hydroxylated metabolites as glucuronides of MTQ instead of the parent drug.

Flunitrazepam excretion patterns using the Abuscreen OnTrak and OnLine immunoassays: comparison with GC-MS.

A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without beta-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with beta-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with beta-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolites.

Autoimmune hemolytic anemia associated with postirradiation malignant stromal tumor (leiomyosarcoma) of the jejunum.

We describe a patient who presented with autoimmune hemolytic anemia and small bowel obstruction secondary to a malignant stromal tumor (leiomyosarcoma) of the jejunum, 25 years postchemotherapy and radiation treatment for stage IIA Hodgkin's disease. The patient was treated with corticosteroid therapy and surgical resection of the jejunal tumor. We conclude that autoimmune hemolytic anemia may be an unusual presentation for postirradiation sarcoma.

Levels of human and rat hypothalamic growth hormone-releasing factor as determined by specific radioimmunoassay systems.

Polyclonal antibodies to synthetic human pancreatic growth hormone-releasing factor [hpGRF(1-44)NH2] and rat hypothalamic growth hormone-releasing factor [rhGRF(1-43)OH] were produced in rabbits by injecting these weak immunogens, coupled to thyroglobulin and emulsified with complete Freund's adjuvant in the presence of activated charcoal, directly into the spleen. A subsequent booster injection by the conventional intramuscular route resulted in high-titer antibodies, which at a 1:20,000 dilution were used to develop highly sensitive and specific radioimmunoassays for these peptides. By using antibodies with an apparent Ka of 3.3 X 10(-12) (human) and 7.7 X 10(-11) (rat), the sensitivity of these assays in both human and rat was found to be less than 1 fmol. The antibody to hpGRF(1-44)NH2 is directed against the COOH-terminal region of the molecule, as shown by its crossreactivity with various hpGRF analogues: 140% with hpGRF(30-44)NH2; 1%-2% with hpGRF(1-37)OH, hpGRF(1-40)OH, and hpGRF(1-40)NH2; and none with hpGRF(1-29)NH2. Serial dilutions of human and rat hypothalamic extracts demonstrated parallelism with the corresponding species-specific standard and 125I-labeled tracer. There was no crossreactivity with other neuropeptides, gastrointestinal peptides, or hypothalamic extracts of other species. The hypothalamic content in fmol/mg (wet weight) of tissue was 3.6 +/- 0.2 for the human and 11.1 +/- 5.5 for the rat. Age-related changes in hypothalamic GRF content were present in rats, with a gradual increase from 2 to 16 weeks and a correlation between increasing body weight and GRF content. These radioimmunoassays will serve as important tools for understanding the regulation of growth hormone secretion in both human and rat.

Sindrome de Poland. Apresentacao de um caso. / Poland's syndrome.Report of a case

Os autores fazem em seu artigo uma revisao de literatura da sindrome de Poland, assim como a descricao de um caso clinico, matriculado em servicos do Hospital Central da Irmandade da Santa Casa de Misericordia de Santos